A representative bioluminescence reaction is the oxidation reaction of a luminescent substrate, “luciferin” (a low-molecular organic compound), catalyzed by an enzyme (protein) called “luciferase.” Luminescence is the release of energy in the form of light (photons), produced when excited oxyluciferin molecules generated immediately after the oxidation reaction of luciferin return to the ground state. Thus, luminescence in which molecules formed by reaction are excited by chemical reaction energy and emit visible light when returning from the excited state to the ground state is called chemiluminescence. In almost all cases, this chemical reaction is an oxidation reaction.
On the other hand, fluorescence is the phenomenon in which a certain kind of substance absorbs the energy of light such as ultraviolet radiation or visible light and emits light. In this process, energy produced when excited molecules return to the ground state by absorption of light energy is released as light (photons). Thus, prompt reemission of excitation energy absorbed by energy-absorbing functional groups (fluorescence chromophores) as light (photons) is fluorescence.
However, the presence of substances having both chemiluminescence activity and fluorescence-generating ability has not yet been known. If such a substance is created, both measurement or detection using chemiluminescence and measurement or detection using fluorescence will become possible in the same molecules. Undoubtedly, that will make a significant contribution to industry.
In addition, chemilumimescent enzymes isolated so far are unstable to heat. For example, when treated at 90° C. for 5 min, they have lost chemiluminescence activity and never been recovered. Development of a heat-resistant chemilumimescent enzyme has been strongly desired. Meanwhile, calcium ion-binding photoproteins react specifically with calcium or strontium ions etc. and emit light instantaneously. Currently, aequorin, clytin, obelin, mitrocomin, mineopsin, bervoin, etc. are known as the calcium ion-binding photoprotein family. Table 1 lists the calcium ion-binding photoproteins whose apoprotein has been isolated.
TABLE 1Species,GeneBankNamescientific nameAcc. No.Authors (year)AequorinAequorea victoriaL29571Inouye et al.(1985)AequorinAequorea victoriaCharbonnueau etal.(1985)AequorinAequorea victoriaM16103Prasher et al.(1987)AequorinAequorea parvaAY013822Luo et al.(2000)AequorinAequorea macrodactylaAY013823Luo et al.(2000)ClytinClytia(=Phialidium)L13247Inouye & Tsuji(=Phialidin)gregarium(1993)MitrocominMitrocoma(=Halistaura)L31623Fagan et al.(=Halistarin)cellularia(1993)ObelinObelia longissimaU07128Illarionov etal.(1995)ObelinObelia geniculataAF394688Markova etal.(2002)
Among the calcium-binding photoproteins, aequorin has been studied especially in detail. Aequorin is a protein complex that binds specifically only with a trace amount of calcium ions and emits light instantaneously. It has been clarified from the crystal structures analyzed with X ray that aequorin is present as a complex (coelenterazine peroxide) composed of apoaequorin (apoprotein), which is the protein portion consisting of 189 amino acids, coelenterazine corresponding to a luminescent substrate, and molecular oxygen (Head, J. F., Inouye, S., Teranishi, K. and Shimomura, O. (2000) Nature, 405, 372-376). The light-emitting reaction and regeneration reaction of aequorin are shown below.

That is, when calcium ions bind to aequorin, a blue light (maximum wave length: 465-470 nm) emission takes place instantaneously; coelenteramid, an oxide of a coelenterazine, dissociates from apoaequorin; and carbon dioxide is released. (Shimomura, O. and Johnson, F. H. (1975) Nature 256, 236-238).
On the other hand, apoaequorin that has bound to calcium ions and emitted light can be regenerated to aequorin having instantaneous light-emitting ability. This regeneration is realized by dissociating calcium ions bound to apoaequorin with a chelating agent such as EDTA and incubating with coelenterazine and oxygen in the presence of a reducing agent (dithiothreitol, 2-mercaptoethanol, etc.) at low temperatures (Shimomura, C. and Johnson, F. H (1970), Nature, 227, 1356-1357).
It has been reported that feeble continuous luminescence is observed in process of the experiment in which natural aequorin is reacted with calcium ions and caused to emit light, and subsequently regenerated to aequorin in the presence of a chelating agent, a reducing agent, and coelenterazine that a luminescent substrate (Shimomura, O. and Johnson, F. H. (1975) Nature 256, 236-238). Further, it has been predicted that a molecular species exhibiting feeble luciferase-like activity would be present in a aequorin solution after having emitted light. However, as to substances involved in feeble luminescence predicted to be present after the light-emitting reaction, participation of complexes etc. of calcium-apoaequorin-coelenteramid, apoaequorin-coelenteramid, and calcium-apoaequorin was not confirmed. In addition, the amount of coelenteramid that is still present after a light-emitting reaction was 17% for natural apoaequorin and 33% for recombinant apoaequorin. That is, although the presence of the complex of calcium ion-apoaequorin-coelenteramid was predicted, it was not isolated and purified, or confirmed. As for the mechanism of feeble continuous luminescence, calcium-apoaequorin-coelenteramid, apoaequorin-coelenteramid, and calcium-apoaequorin have not been isolated or identified, either. The presence of these complexes was not predicted based on the precise fact, and was only speculated (Shimomura, O. (1995) Biochem. J. 306, 537-543).
On the other hand, the inventors have already reported that the mere addition of coelenterazine to calcium-bound apoaequorin (calcium-apoaequorin) without coelenteramid exhibits feeble continuous luminescence (Japanese Laid-Open Application No. 1989-47379). However, it has not been known what kind of substance it is that emits feeble luminescence.
An object of the present invention is to provide a novel luminescent substance and a novel fluorescent substance. It turned out, surprisingly, that the substances produced for that purpose are the first that have both chemiluminescence activity and fluorescence-generating ability.
Accordingly, the first object of the present invention is to provide a fluorescent protein having chemiluminescence activity. Specifically, it is to provide a novel fluorescent protein (bFP) having chemiluminescence activity, generated from a calcium-binding photoprotein, and further, a method for producing such a protein together with its specific use.
The second object is to produce another novel fluorescent protein (gFP) from the fluorescent protein (bFP) having chemiluminescence activity and to provide its specific use.